Yanovsky-Dagan Shira, Frumkin Ayala, Lupski James R, Harel Tamar
Department of Genetics, Hadassah Medical Organization, Jerusalem, Israel.
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
HGG Adv. 2022 Jan 25;3(2):100092. doi: 10.1016/j.xhgg.2022.100092. eCollection 2022 Apr 14.
Paralogs and pseudogenes are abundant within the human genome, and can mediate non-allelic homologous recombination (NAHR) or gene conversion events. The locus contains three paralogs situated in tandem, and is therefore prone to NAHR-mediated deletions and duplications associated with severe neurological phenotypes. To study this locus further, we aimed to generate biallelic loss-of-function variants in by CRISPR/Cas9 genome editing. Unexpectedly, two of the generated clones underwent gene conversion, as evidenced by replacement of the targeted sequence of by a donor sequence from its paralog We highlight the complexity of CRISPR/Cas9 design, end-product formation, and recombination repair mechanisms for CRISPR/Cas9 delivery as a nucleic acid molecular therapy when targeting genes that have paralogs or pseudogenes, and advocate meticulous evaluation of resultant clones in model organisms. In addition, we suggest that endogenous gene conversion may be used to repair missense variants in genes with paralogs or pseudogenes.
旁系同源基因和假基因在人类基因组中大量存在,并可介导非等位基因同源重组(NAHR)或基因转换事件。该基因座包含三个串联排列的旁系同源基因,因此容易发生与严重神经学表型相关的NAHR介导的缺失和重复。为了进一步研究该基因座,我们旨在通过CRISPR/Cas9基因组编辑在其中产生双等位基因功能丧失变体。出乎意料的是,两个产生的克隆发生了基因转换,这通过其旁系同源基因的供体序列取代该基因座的靶向序列得到证明。我们强调了在靶向具有旁系同源基因或假基因的基因时,作为核酸分子疗法的CRISPR/Cas9设计、终产物形成和重组修复机制的复杂性,并主张在模式生物中对所得克隆进行细致评估。此外,我们建议内源性基因转换可用于修复具有旁系同源基因或假基因的基因中的错义变体。